Penicillin-polyvinyl alcohol conjugate and process of preparation

ABSTRACT

This invention relates to tolerogenic conjugates, suitable for use in suppressing the formation of reaginic antibodies in a host, of haptenic determinants, in particular the benzylpenicilloyl (BPO) group and 2,4-dinitrophenyl (DNP) group, with water-soluble, nonimmunogenic polymers, i.e. polyvinyl alcohols (PVA). The invention also comprises a method for the preparation of the conjugate and a method of suppressing the formation of these antibodies in a mammal sensitive to penicillin.

This is a division of application Ser No. 15,864, filed Feb. 27, 1979, now U.S. Pat. No. 4,296,097 and the benefits of 35 USC 120 are claimed relative to it.

The present invention relates to a conjugate for use for the suppression of reaginic antibodies in a person allergic to penicillin and to a method for making the conjugate.

One to 5% of the population in developed countries are allergic to penicillin. When penicillin is administered, they may go into anaphylactic shock or develop rashes or various other symptoms of penicillin hypersensitivity. The various allergic reactions to penicillin are mediated by different types of antibodies, the reactions of the "immediate type" being due to reaginic antibodies known as IgE antibodies. The benzylpenicilloyl (BPO) group has been shown to be the major immunogenic determinant in penicillin hypersensitivity.

The formation of anti-BPO IgE antibodies is primarily triggered by the haptenic BPO group. As any other hapten, BPO by itself does not induce the formation of IgE, but does so when conjugated with either (i) macromolecular contaminants or additives present in the penicillin preparation, or (ii) with the patient's own proteins or tissue components. The BPO conjugate(s) stimulates in the body appropriate lymphoid cells to produce anti-BPO IgE antibodies. These antibodies become attached to mast cells and basophils. Upon subsequent administration of penicillin, the polyvalent BPO conjugates (present in the penicillin preparation or formed by reaction with the patient's proteins) become attached to the anti-BPO IgE antibodies fixed to the mast cells or basophils. This antibody-antigen interaction results in bridging (i.e. crosslinking) of the cell fixed anti-BPO IgE antibodies and appears to be the prerequisite step for inducing the release from these cells of the granules containing histamine and other pharmacologically vasoactive compounds, which react with appropriate target organs leading thus to the inflammatory reactions underlying allergic manifestations.

Various methods have been used for the treatment of such allergic reactions; one pharmacological method involves the administration of antihistamines. Another method, based on immunochemical principles, involves the administration of a univalent BPO derivative, i.e. benzylpenicilloyl-formyl lysine (BPO-FLYS), which was synthesized by A. L. de Weck of Bern, Switzerland, and which is incapable of coupling to the host's constituents to form polyvalent allergens.* Such derivatives can attach themselves to each of the antigen binding sites of the individual IgE antibodies fixed to the mast cells or basophils, thereby blocking (preventing) the antigen binding sites of these antibodies for subsequent attachment to polyvalent molecules, i.e. thus, the possibility of triggering the release of granules from the cells is prevented. However, neither of these treatments prevents the formation of more IgE antibodies upon further exposure to the allergen.

In the novel method described here by the inventors, it was discovered that it is possible to suppress the formation of anti-BPO IgE reaginic antibodies by a judicious choice of a conjugate of BPO with a nonimmunogenic carrier, consisting of polyvinyl alcohols (PVA). Thus, treatment of either normal animals or of animals sensitized to BPO with a compound consisting of BPO attached to PVA was shown to induce immunological tolerance.

Immunological tolerance may be defined as a specific inhibition of the immune response to a given antigen. Thus, under certain conditions, the organism, instead of developing an immune response on exposure to a given antigen, may exhibit a state of immunological paralysis to the antigen in question, i.e. the administration of the particular antigen does not induce an immune response. However, the immune response of the host to other unrelated antigens remains normal. This state of immunologically specific tolerance is also known as immune paralysis and the substance capable of inducing tolerance, rather than antibody production, is referred to as a tolerogen. Clearly, the term tolerogenic is not synonymous with nonimmunogenic; the latter term indicates simply that a given compound is immunologically inert, i.e. it plays a passive role in relation to an immune response and is, therefore, not capable of triggering an immune response. By contrast, the term tolerogenic implies an active role for the corresponding antigen in suppressing, i.e. in shutting off, the immune response with respect to itself and related compounds.

In a paper published in the Journal of Immunology, Vol. 117, No. 3, Sept. 1976, pp. 927-934, the present inventors reported a regimen for the suppression of the formation of antibodies of the IgE class to the BPO group in experimental mice by administration of conjugates of BPO with the nonimmunogenic carrier, consisting of isologous γ-globulins, i.e. γ-globulins of murine origin. Similarly, the use of a synthetic polypeptide, i.e. the copolymer of D-glutamic acid and D-lysine (D-GL) as a carrier for BPO groups for the suppression of anti-BPO IgE response was also successful, as reported by Chiorazzi et al. (Chiorazzi, N., Eshhar, Z. and Katz, D. H., Proc. Nat. Acad. Sci. USA, 73:2091 [1976]). As described in the present submission, the inventors have discovered a new and more effective tolerogenic conjugate, which was prepared by coupling BPO to PVA. The resulting BPO-PVA conjugates were shown to suppress the anti-BPO IgE response in either normal or sensitized mice. In a parallel study, it was also shown that IgE antibodies directed against the 2,4-dinitrophenyl (DNP) group were also suppressed on administration of DNP-PVA conjugates. Accordingly, the present invention comprises conjugates suitable for suppressing the formation of reaginic antibodies in a host to small haptenic groups, e.g. BPO and DNP, by injection of the conjugates of these groups with the nonimmunogenic, water-soluble polymer, namely PVA.

In the particular case of the tolerogenic conjugates prepared in this study, these conjugates contained an average of 0.7-3.9 BPO or DNP groups per molecule of PVA of molecular weight in the range of 10,000-14,000.

This invention comprises also a process for activating PVA with cyanogen bromide, followed by reaction with 1,6-hexane diamine so as to graft amino groups onto the PVA molecules. Penicillin was then reacted with the amino derivatives of PVA at alkaline pH.

In addition, the invention comprises a process for the suppression of the formation of anti-BPO reaginic antibodies in a mammal sensitive to penicillin by injecting the conjugate into the sensitized individual in a pharmaceutically acceptable manner.

The preferred nonimmunogenic, tolerogenic carriers are polyvinyl alcohols of molecular weights of 10,000 and 14,000.

The reaction of penicillin with the amino derivative of PVA is carried out in the presence of a suitable salt which permits the reaction to proceed at a pH of about 9.8. Suitable salts include potassium carbonate and sodium carbonate.

The present invention will be illustrated in the following nonlimiting Examples and Tables.

MATERIALS AND METHODS Animals

Inbred 8- to 12-week old (C₅₇ BL/6×DBA/2)F₁ mice (designated as B₆ D₂ F₁) were purchased from Jackson Laboratories, Bar Harbor, Maine. For the passive cutaneous anaphylaxis (PCA) assays, random-bred hooded rats, supplied by Bio-Breeding Laboratories Ltd., Ottawa, were used.

Preparation of amino derivative of PVA (PVA-NH₂)

For the sake of brevity, PVA of molecular weights of 10,000 and 14,000 will be referred to hereafter as PVA₁₀ and PVA₁₄, respectively. To PVA₁₀ or PVA₁₄ (10 gm) dissolved in 200 ml of distilled water was added 5 gm by cyanogen bromide. The pH of the reaction mixture was maintained at 11 by dropwise addition of 2 N NaOH. Twenty minutes later, 10 gm of 1,6-hexane diamine was added to the mixture and stirred for 48 hours at room temperature. The reaction mixture was then dialyzed exhaustively against distilled water and lyophilized.

Preparation of BPO-PVA

PVA-NH₂ (400 mg) was dissolved in 20 ml distilled water. After addition of 400 mg of penicillin G (potassium salt, Pfizer Company Ltd., Montreal) and 4 ml of 1 M K₂ CO₃, the mixture was stirred for 21/2 hours at room temperature. The unreacted penicillin G was removed by gel filtration on Sephadex G-25 column. The sample was dialyzed and lyophilized. The degree of substitution, i.e. the number of BPO groups coupled per PVA molecule, was determined by quantitative titration with mercuric chloride solution according to the method of Parker et al. (Journal of Experimental Medicine, Vol. 115, p. 803, 1962) and found to be 2.8. The other BPO_(x) -PVA conjugates of varying epitope density prepared in this study, i.e. PBO₀.7 -PVA₁₀, BPO₁.1 -PVA₁₀, BPO₂ -PVA₁₀, BPO₂.7 -PVA₁₀, BPO₂.8 -PVA₁₀, BPO₂.7 -PVA₁₄ and BPO₃.9 -PVA₁₄, were prepared in a similar fashion by adjusting appropriately the concentration of penicillin G and allowing the reaction to proceed for different times.

Preparation of DNP-PVA

To a solution of 50 ml of 0.4 M Na₂ CO₃ containing 0.5 gm of PVA-NH₂ was added 0.5 gm of sodium 2,4-dinitrobenzene sulfonate (Baker Chemical Co., Phillipsburg, N.Y.) and the mixture was incubated at 37° C. for 3 days. The precipitate, which formed during the incubation period, was removed by centrifugation at 18,000 rpm in a Sorvall RC2-B centrifuge for 20 min. The supernatant was passed through a column (4×60 cm) of Sephadex G-25 gel to eliminate any unreacted hapten. The effluent containing the conjugate was dialyzed against distilled water and lyophilized. The degree of substitution of DNP groups, i.e. the average number of DNP groups grafted onto a PVA molecule, was determined spectrophotometrically taking the molar absorption coefficient for DNP as 17,400 at 360 nm, and was found to be 1.5.

The precipitate collected by centrifugation as described above was dissolved in distilled water, dialyzed exhaustively and lyophilized. The degree of substitution of this sample was found to be 2.4.

The conjugate DNP₃.1 -PVA₁₀ was similarly prepared except that the Na₂ CO₃ concentration was lowered to 0.2 M and the reaction mixture was stirred for 2 days at room temperature. The conjugate DNP₄.3 -PVA₁₄ was prepared as DNP₃.1 -PVA₁₀ with the exception that 0.75 gm of PVA₁₄ -NH₂ was used in the reaction mixture.

Preparation of hapten-protein conjugates

The benzylpenicilloyl-ovalbumin (BPO₄ -OA) and benzylpenicilloyl-bovine γ-globulin (BPO₁₇ -BGG) conjugates were prepared as described in a previous study (Lee, W. Y. and Sehon, A. H., J. Immunol. 117:927 [1976]) and the DNP₃ -OA and DNP₁₈ -BGG conjugates were synthesized as reported earlier (Lee, W. Y. and Sehon, A. H., J. Immunol. 114:829 [1975]). The 2,4-dinitrophenylated extract of Ascaris suum (DNP-ASC), which contained 6.5×10⁻⁸ M DNP per mg of ASC, was prepared by reacting 46 mg of ASC with 24 mg of DNBS in the presence of 46 mg of Na₂ CO₃ in a total volume of 5.5 ml of distilled water at 37° C. for 2 hours.

Immunization and measurement of reaginic antibody responses

For the optimal elicitation of anti-DNP and anti-OA IgE responses, mice were injected i.p. with the standard, low dose of 1 μg of DNP₃ -OA suspended with 1 mg of freshly prepared aluminum hydroxide in 0.5 ml of phosphate buffered saline (PBS). This dose, when administered by the i.p. route, will be referred to hereafter as the sensitizing dose of DNP₃ -OA.

The optimal anti-BPO IgE response was induced in mice by i.p. injection of 10 μg of BPO₄ -OA in presence of 1 mg of Al(OH)₃. Mice were also sensitized with 10 μg of DNP-ASC premixed with 1 mg of Al(OH)₃ in 0.5 ml of PBS for the induction of anti-DNP and anti-ASC IgE antibodies. Groups of four to five mice were treated in an identical manner and the sera of mice within each group were pooled for determination of the average reaginic titer by the passive cutaneous anaphylaxis (PCA) assay. The end point of the titration was taken as the reciprocal of the highest dilution of each serum resulting in a reaction of 5 mm in diameter. The PCA titers are reported as averages of two determinations.

Adoptive cell transfer

This procedure consisted of the transfer of spleen cells from sensitized and/or tolerized mice into X-irradiated (650 R) syngeneic recipients and injection of the recipients with the standard sensitizing dose of antigen in presence of Al(OH)₃ within 4 hours after cell transfer.

EXAMPLE 1: SUPPRESSION OF IgE RESPONSE SPECIFIC FOR BENZYLPENICILLOYL GROUP (A) Suppression of the Induction of Anti-BPO IgE Response with BPO₂ -PVA₁₀ or BPO₂.7 -PVA₁₄

Mice were injected i.p with BPO₂ -PVA₁₀ or BPO₂.7 -PVA₁₄, in doses of 0.5 or 1.0 mg per recipient, 7 days prior to sensitization with BPO₄ -OA in presence of Al(OH)₃. The primary IgE responses were determined 14 days after sensitization. For the induction of the secondary and tertiary IgE responses, all mice were reimmunized with sensitizing doses of BPO₄ -OA on days 28 and 56, respectively, and the PCA titers were determined 7 days after sensitization. The results indicated in Table I clearly show that the anti-BPO IgE response was profoundly suppressed in spite of repeated challenge of these animals with the sensitizing dose of the antigen without, however, affecting appreciably the anti-OA IgE response.

(B) Suppression of the Anti-BPO IgE Response in Sensitized Animals with BPO₂ -PVA₁₀

The ability of BPO₂ -PVA₁₀ to abrogate an ongoing anti-BPO IgE response was tested in sensitized animals as illustrated in Table II. All mice received sensitizing doses of BPO₄ -OA on days 0, 28 and 56. Mice in test groups received additional injections of 0.5, 0.75 or 1.0 mg of BPO₂ -PVA₁₀ 21 days after primary sensitization of mice. As is evident from the PCA results determined with sera obtained on day 35, which are listed in Table II, the ongoing anti-BPO IgE response was completely abolished after injection of the tolerogen 7 days before the secondary sensitization. Moreover, as supported by the PCA titers of sera of mice which received a third sensitizing dose of BPO₄ -OA, these mice had remained immunosuppressed for at least 36 days after receiving an injection of the tolerogenic BPO₂ -PVA₁₀ preparation.

(C) Effect of Dose of BPO₂.7 -PVA₁₄ on the Abrogation of the Anti-BPO Response in Sensitized Mice

Various doses ranging from 0.1 to 1.0 mg of BPO₂.7 -PVA₁₄ were injected into mice which had been sensitized 21 days earlier. As indicated in Table III, a single dose of 0.25 mg of BPO₂.7 -PVA₁₄ was sufficient to abolish completely an ongoing anti-BPO IgE response.

(D) Tolerogenicity of BPO_(x) -PVA₁₀ Conjugates with Different Epitope Density in Sensitized Mice

The effect of different epitope density (i.e. degree of substitution) on the tolerogenicity of conjugates of BPO with PVA was studied by injecting 1 mg of these conjugates i.p. into mice which had been sensitized with BPO₄ -OA suspended in Al(OH)₃ 21 days earlier. One week after injection of the tolerogen, all mice were immunized with a sensitizing dose of BPO₄ -OA and the PCA titers were determined 7 days later. The results shown in Table IV demonstrate that complete suppression of anti-BPO IgE response was achieved by injection of 1 mg of BPO_(x) -PVA₁₀ conjugate with an epitope density in excess of 1.1 and that conjugates of lower epitope density, e.g. BPO₀.7 -PVA₁₀, suppressed only partially the anti-BPO IgE response. It is to be stressed that the suppression of anti-BPO IgE response was antigen specific, since the accompanying anti-OA IgE response was not affected.

EXAMPLE 2: SUPPRESSION OF IgE RESPONSE SPECIFIC FOR DNP GROUP (A) Suppression of Anti-DNP IgE Response with DNP₃.4 -PVA₁₀

The suppression of the induction of anti-DNP IgE response was studied by injecting a single dose of 1 mg of DNP₃.4 -PVA₁₀ into normal mice one week prior to sensitization with the standard dose of 1 μg of DNP₃ -OA in presence of 1 mg of Al(OH)₃. The results plotted in FIG. 1 show that the anti-DNP IgE response was profoundly suppressed in mice treated with this conjugate. The suppression was antigen specific as demonstrated by the fact that the anti-OA IgE response was virtually unaffected.

(B) Abrogation of an Ongoing Anti-DNP IgE Response with DNP₃.4 -PVA₁₀

The possibility of using the above immunosuppressive approach for the abrogation of an ongoing anti-DNP IgE response was explored by injection of 1 mg of DNP₃.4 -PVA₁₀ into mice which had been sensitized with DNP₃ -OA 14 days earlier as shown in FIG. 2. It is clear that the anti-DNP IgE response was abrogated readily after injection of the tolerogen. However, these mice produced high level of IgE response to OA which was indistinguishable from those produced by the control mice.

(C) Effect of Dose of DNP₄.3 -PVA₁₄ on Suppression of Anti-DNP IgE Response

Mice were injected i.p. with varying doses of the tolerogen 7 days prior to sensitization with DNP₃ -OA in presence of Al(OH)₃. From the results listed in Table V, it is evident that a significant degree of suppression of the anti-DNP IgE response was achieved with all the doses tested and that the higher doses were more effective in inducing immunosuppresion. Upon challenge with a secondary sensitizing dose of DNP₃ -OA on day 28, the mice which had received the tolerogen exhibited almost total immunosuppression or their anti-DNP IgE response remained markedly dampened.

(D) Effect of time of Injection of DNP₄.3 -PVA₁₄ on Reaginic Antibody Formation

Mice were injected i.p. with the tolerogen (1 mg per mouse) on various days before or after immunization with DNP-OA in Al(OH)₃. Sera were collected at the peak of reaginic antibody response and assayed for PCA activities as indicated in Table VI. It is obvious that the anti-DNP IgE response could be readily suppressed regardless of the time of injection. Moreover, the immunosuppression was more profound when the tolerogen was injected close to time of immunization, i.e. from day -7 to day 4.

(E) Abrogation of Anti-DNP IgE Response with DNP₂.4 -PVA₁₀ in Sensitized Mice

For the abrogation of an ongoing anti-DNP IgE response, mice which had been sensitized with DNP₃ -OA 59 days earlier were injected with 1 mg of DNP₂.4 -PVA₁₀ and resensitized on the following day. As is evident from Table VII, the anti-DNP response was suppressed significantly. To test the duration of the unresponsive state, these mice received two additional sensitizing doses of DNP₃ -OA at 4-week intervals. It is obvious from Table VII that the unresponsive state was long lasting despite the administration of repeated sensitizing injections of DNP₃ -OA.

(F) Specificity of Immunosuppression by DNP₂.4 -PVA₁₀

It has been shown in previous sections that injection of mice with DNP-PVA conjugates suppressed specifically the anti-DNP response. Hence, this experiment was designed to examine the effect of DNP-PVA on the anti-DNP IgE response induced by DNP conjugates with different carriers. For this purpose, mice were injected with 1 mg of DNP₂.4 -PVA₁₀ two days before sensitization with a standard dose of DNP₃ -OA (Expt. 1) or DNP-ASC (Expt. 2), followed by resensitization 4 weeks later with a DNP conjugate with different carrier, namely DNP-ASC (Expt. 1) or DNP-OA (Expt. 2), as indicated in Table VIII. The results demonstrated that the anti-DNP IgE response was effectively suppressed regardless of the nature of the carrier used for sensitization.

(C) Active Suppression of Anti-DNP IgE Response of Primed Cells with Tolerized Cells in Adoptive Transfer System

In order to study the possibility of induction of suppressor cells by the treatment with DNP-PVA, the adoptive cell transfer system was used. Different combinations of spleen cells from primed and tolerized mice were transferred into X-irradiated (650 R) recipients. The primed donors were sensitized with DNP₃ -OA 30 days before the cell transfer; the tolerized donors received an additional injection of 1 mg of DNP₂.4 -PVA₁₀ 7 days prior to cell transfer. The results illustrated in FIG. 3 clearly show that the tolerized cells remained unresponsive in the X-irradiated recipients. Moreover, these tolerized cells suppressed actively the anti-DNP IgE response of primed cells in the cell mixture. This finding indicates that DNP-specific suppressor cells are present in the tolerized cell populations.

(H) Maintenance of Unresponsiveness in Adoptive Cell Transfer System

The donor mice were presensitized with a sensitizing dose of DNP₃ -OA 49 days before the cell transfer. As indicated in Table IX, at various times prior to cell transfer, the donors received an additional i.p. injection of 1 mg of DNP₂.4 -PVA₁₀. The spleen cells of these donors (3×10⁷) were transferred into X-irradiated (650 R) recipients which received a sensitizing dose of DNP₃ -OA within 4 hours after cell transfer. The results shown in Table IX demonstrate that the spleen cells from donors tolerized 5 hours or 1 day prior to transfer did not show significant suppression in the X-irradiated recipients. On the other hand, the spleen cells of donors which had received DNP₂.4 -PVA₁₀ 2 days to 14 days prior to cell transfer remained unresponsive in the X-irradiated recipients. This implies that treatment of mice with DNP₂.4 -PVA₁₀ requires at least 2 days to render the cells unresponsive.

(I) Prevention of Systemic Anaphylaxis by Treatment of Sensitized Mice with DNP₂.4 -PVA₁₀

Since it has been shown in previous sections that an ongoing anti-DNP IgE response could be readily abrogated by treatment of sensitized mice with DNP-PVA conjugates, the present experiment was designed to examine if systemic reactions could occur in these immunosuppressed animals when they were challenged i.v. with a multivalent DNP-protein conjugate (i.e. DNP₉ -MγG) or OA. In order to render the mice sensitive for the demonstration of systemic anaphylaxis, the administration of B. pertussis in addition to the sensitizing antigen was found to be essential. As indicated in Table X, treatment of mice with 1 mg of DNP₂.4 -PVA₁₀ suppressed completely the anti-DNP IgE response, whereas the anti-carrier IgE response was not affected. More importantly, whereas the control sensitized mice died of anaphylaxis after i.v. challenge of DNP₉ -MγG, the mice rendered tolerant by this treatment did not show any discomfort on i.v. challenge with DNP₉ -MγG. Moreover, i.v. challenge with OA led to the death of both groups of mice indicating, thus, that the immunosuppression was highly antigen specific.

                                      TABLE I                                      __________________________________________________________________________     SUPPRESSION OF THE INDUCTION OF ANTI-BPO IgE RESPONSE WITH                     BPO.sub.2 --PVA.sub.10 OR BPO.sub.2.7 --PVA.sub.14                                            PCA TITERS                                                                     PRIMARY     SECONDARY   TERTIARY                                TOLEROGEN INJECTED*                                                                           ANTI-BPO                                                                             ANTI-OA                                                                              ANTI-BPO                                                                             ANTI-OA                                                                              ANTI-BPO                                                                             ANTI-OA                           __________________________________________________________________________     BPO.sub.2 --PVA.sub.10, 0.5 mg                                                                <10   1,450  20   3,160 1,260 5,370                             BPO.sub.2 --PVA.sub.10, 1.0 mg                                                                <10   1,580 <10   2.820 360   4,360                             BPO.sub.2.7 --PVA.sub.14, 0.5 mg                                                              <10   1,740  10   3,980 360   6,030                             BPO.sub.2.7 --PVA.sub.14, 1.0 mg                                                              <10     680 <10   3,020 320   5,620                             CONTROL MICE    30   1,910  810  3,980 2,750 6,920                             __________________________________________________________________________      *Mice were injected i.p. with BPO.sub.2 --PVA.sub.10 or BPO.sub.2.7            --PVA.sub.14 7 days prior to sensitization with BPO.sub.4 --OA in presenc      of Al(OH).sub.3. The primary IgE responses were determined 14 days after       sensitization. For the induction of secondary and tertiary IgE responses,      all mice were immunized with sensitizing doses of BPO.sub.4 --OA on days       28 and 56, respectively, and the PCA titers were determined 7 days after       sensitization.                                                           

                  TABLE II                                                         ______________________________________                                         ABROGATION OF ANTI-BPO IgE RESPONSE WITH                                       BPO.sub.2 --PVA.sub.10 IN SENSITIZED ANIMALS                                                      PCA TITERS                                                         TREATMENT*            ANTI-   ANTI-                                     GROUP    DAY     COMPOUND    DAY   BPO   OA                                    ______________________________________                                         CONTROL  0       BPO.sub.4 --OA                                                                             14     90   3,100                                          28      BPO.sub.4 --OA                                                                             35     870  3,630                                          56      PBO.sub.4 --OA                                                                             63    1,450 4,170                                 TEST 1   0       BPO.sub.4 --OA                                                                             14     90   3,100                                          21      BPO.sub.2 --PVA.sub.10,                                                        0.5 mg                                                                 28      BPO.sub.4 --OA                                                                             35    <10   5,370                                          56      BPO.sub.4 --OA                                                                             63     930  6,920                                 TEST 2   0       BPO.sub.4 --OA                                                                             14     90   3,100                                          21      BPO.sub.2 --PVA.sub.10,                                                        0.75 mg                                                                        BPO.sub.4 --OA                                                                             35    <10   3,240                                          56      BPO.sub.4 --OA                                                                             63    <10   5,370                                 TEST 3   0       BPO.sub.4 --OA                                                                             14     90   3,100                                          21      BPO.sub.2 --PVA.sub.10,                                                        1.0 mg                                                                 28      BPO.sub.4 --OA                                                                             35    <10   4,470                                          56      BPO.sub.4 --OA                                                                             63     360  6,610                                 ______________________________________                                          *All mice received sensitizing doses of BPO.sub.4 --OA on days 0, 28 and       56. Mice in the test groups received additional injections of 0.5 mg (TES      1), 0.75 mg (TEST 2) or 1.0 mg (TEST 3) of BPO.sub.2 --PVA.sub.10 on day       21.                                                                      

                  TABLE III                                                        ______________________________________                                         EFFECT OF DOSE OF BPO.sub.2.7 --PVA.sub.14 ON                                  THE ABROGATION OF ANTI-BPO RESPONSE IN                                         SENSTIZED MICE                                                                                    PCA TITERS                                                  TREATMENT*                   ANTI-   ANTI-                                     GROUP   DAY     COMPOUND     DAY   BPO   OA                                    ______________________________________                                         CON-    0       BPO.sub.4 --OA                                                                              14    70    4,000                                 TROL    28      BPO.sub.4 --OA                                                                              35    1,050 4,570                                         56      BPO.sub.4 --OA                                                                              63    1,350 3,630                                 TEST 1  0       BPO.sub.4 --OA                                                                              14    70    4,000                                         21      BPO.sub.2.7 --PVA.sub.14,                                                      0.1 mg                                                                 28      BPO.sub.4 --OA                                                                              35    20    5,750                                         56      BPO.sub.4 --OA                                                                              63    790   4,470                                 TEST 2  0       BPO.sub.4 --OA                                                                              14    70    4,000                                         21      BPO.sub.2.7 --PVA.sub.14,                                                      0.25 mg                                                                28      BPO.sub.4 --OA                                                                              35    <10   3,800                                         56      BPO.sub.4 --OA                                                                              63    100   3,380                                 TEST 3  0       BPO.sub.4 --OA                                                                              14    70    4,000                                         21      BPO.sub.2.7 --PVA.sub.14,                                                      0.5 mg                                                                 28      BPO.sub.4 --OA                                                                              35    <10   5,370                                         56      BPO.sub.4 --OA                                                                              63    90    2,950                                 TEST 4  0       BPO.sub.4 --OA                                                                              14    70    4,000                                         21      BPO.sub.2.7 --PVA.sub.14,                                                      1.0 mg                                                                 28      BPO.sub.4 --OA                                                                              35    <10   5,750                                         56      BPO.sub.4 --OA                                                                              63    100   2,560                                 ______________________________________                                          *All mice received sensitizing doses of BPO.sub.4 --OA on days 0, 28 and       56. Mice in the test groups received additional injections of various          doses of BPO.sub.2.7 --PVA.sub.14 on day 21.                             

                  TABLE IV                                                         ______________________________________                                         TOLEROGENICITY OF BPO.sub.x --PVA.sub.10 CONJUGATES                            WITH DIFFERENT EPITOPE DENSITY IN                                              SENSITIZED MICE                                                                               PCA TITERS                                                      TREATMENT*       ANTI-BPO  ANTI-OA                                             ______________________________________                                         BPO.sub.0.7 --PVA.sub.10                                                                         280      6,170                                               BPO.sub.1.1 --PVA.sub.10                                                                        <10       3,800                                               BPO.sub.2.0 -PVA.sub.10                                                                         <10       3,550                                               BPO.sub.2.7 --PVA.sub.10                                                                        <10       5,750                                               BPO.sub.2.8 --PVA.sub.10                                                                        <10       3,980                                               BPO.sub.3.9 --PVA.sub.14                                                                        <10       5,120                                               CONTROL MICE     1,050     4,570                                               ______________________________________                                          *All mice were presensitized with BPO.sub.4 --OA 21 days prior to i.p.         injections of 1 mg of BPO.sub.x --PVA.sub.10 conjugates of different           epitope density. One week after the injection of BPO.sub.x --PVA.sub.10,       mice were sensitized with BPO.sub.4 --OA and the PCA titers determined 7       days later.                                                              

                  TABLE V                                                          ______________________________________                                         EFFECT OF DOSE OF DNP.sub.4.3 --PVA.sub.14 ON SUPPRESSION                      OF ANTI-DNP IgE RESPONSES                                                                    PCA TITERS                                                                     PRIMARY   SECONDARY                                                             ANTI-    ANTI-   ANTI- ANTI-                                    DOSE OF TOLEROGEN*                                                                            DNP      OA      DNP   OA                                       ______________________________________                                         0.25 mg         90      830     330   3,310                                    0.5 mg          80      1,280   150   1,450                                    0.75 mg         20      510     70    1,120                                    1.0 mg          10      380     70    2,460                                    1.5 mg          20      1,410   40    3,160                                    2.0 mg          <10     160     10    2,460                                    CONTROL MICE    380     500     870   1,180                                    ______________________________________                                          *Mice were injected i.p. with varying doses of the tolerogen 7 days prior      to sensitization with DNP.sub.3 --OA in presence of Al(OH).sub.3. The          primary IgE responses were determined on day 14 after sensitization. All       mice were sensitized again on day 28 for the secondary responses. The          secondary antiDNP and antiOA IgE responses were measured 7 days after          immunization.                                                            

                  TABLE VI                                                         ______________________________________                                         EFFECT OF TIME OF INJECTION OF DNP.sub.4.3 --PVA.sub.14 ON                     REAGINIC ANTIBODY FORMATION                                                              PCA TITERS                                                                     PRIMARY      SECONDARY                                               TIME OF     ANTI-              ANTI-  ANTI-                                    TREATMENT*  DNP      ANTI-OA   DNP    OA                                       ______________________________________                                         Day -14     90       1,580     70     3,160                                    DAY -10     210      1,580     220    3,800                                    DAY -7      30       1,280     150    2,950                                    DAY -3      30       1,320     30     2,400                                    DAY -1      50       1,280     50     2,630                                    DAY 0       30       1,280     20     4,170                                    DAY 1       90       1,510     80     6,610                                    DAY 4       70       1,380     90     3,980                                    DAY 7       190      2,340     330    5,750                                    DAY 14      (1,050     1,320)**                                                                               70     5,890                                    DAY 21      (1,050     1,320)**                                                                               180    6,460                                    CONTROL MICE                                                                               1,050    1,320     1,510  3,090                                    ______________________________________                                          *Animals were treated with the tolerogen on the same day (i.e. day 0), or      before, or after i.p. immunization with DNP.sub.3 --OA in Al(OH).sub.3.        The primary IgE responses were determined on day 14 after immunization.        For the induction of secondary responses, all mice were sensitized with        DNP.sub.3 --OA on day 28 and the PCA titers were determined 7 days later.      **These PCA titers represent the primary responses prior to the injection      of tolerogen.                                                            

                                      TABLE VII                                    __________________________________________________________________________     ABROGATION OF ANTI-DNP IgE RESPONSE WITH                                       DNP.sub.2.4 PVA.sub.10 IN SENSITIZED ANIMALS                                    ##STR1##                                                                             TREATMENT*     PCA TITERS                                               GROUP  DAY COMPOUND   DAY ANTI-DNP                                                                              ANTI-OA                                       __________________________________________________________________________     CONTROL                                                                               0   DNP.sub.3OA                                                                                 7 3,020  7,940                                                               14  3,320  3,800                                                28  DNP.sub.3OA                                                                               35  3,020  6,840                                                               42  3,020  6,760                                                56  DNP.sub.3OA                                                                               63  3,020  5,750                                                               70  2,280  4,820                                         TEST   -1  DNP.sub.2.4PVA.sub.10, 1 mg                                                0   DNP.sub.3OA                                                                                7    260  6,840                                                               14    350  3,470                                                28  DNP.sub.3OA                                                                               35    230  9,120                                                               42    260  6,760                                                56  DNP.sub.3OA                                                                               63    390  6,760                                                               70    180  5,490                                         __________________________________________________________________________      *All mice, which had been sensitized with 1 μg DNP.sub.3OA in the           presence of 1 mg Al(OH).sub.3 60 days before the experiment, received          three sensitizing doses of DNP.sub.3OA on days 0, 28 and 56. Mice in test      group received an additional i.p. injection of 1 mg DNP.sub.2.4PVA.sub.10      on day -1.                                                               

                                      TABLE VIII                                   __________________________________________________________________________     SPECIFICITY OF IMMUNOSUPPRESSION BY DNP.sub.2.4 --PVA.sub.10                            TREATMENT*   PCA TITERS                                               GROUP    DAY COMPOUND DAY ANTI-DNP                                                                              ANTI-OA                                                                              ANTI-ASC                                __________________________________________________________________________     EXPERIMENT 1                                                                   CONTROL  -2  SALINE                                                                     0   DNP.sub.3 --OA                                                                          7   <10    <10   N.T.**                                                        14  1,700  1,820 N.T.                                             28  DNP--ASC 35  3,470  3,160 20                                                            42  2,690  830   70                                      TEST     -2  DNP.sub.2.4 --PVA.sub.10                                                   0   DNP.sub.3 --OA                                                                          7   <10    <10   N.T.                                                          14  20     1,740 N.T.                                             28  DNP--ASC 35  <10    1,700 10                                                            42  <10    760   80                                      EXPERIMENT 2                                                                   CONTROL  -2  SALINE                                                                     0   DNP--ASC 7   <10    N.T.  <10                                                           14  300    N.T.  100                                              28  DNP.sub.3 --OA                                                                          35  3,540  80    370                                                           42  3,500  480   320                                     TEST     -2  DNP.sub.2.4 --PVA.sub.10                                                   0   DNP--ASC 7   <10    N.T.  <10                                                           14  <10    N.T.  80                                               28  DNP.sub.3 --OA                                                                          35  <10    20    240                                                           42  20     810   80                                      __________________________________________________________________________      *In experiment 1, all mice received sensitizing doses of DNP.sub.3 --OA        and DNP--ASC on days 0 and 28, respectively. The test mice received            additional injections of 1 mg of DNP.sub.2.4 --PVA.sub.10 on day -2.           In experiment 2, all mice were immunized with sensitizing doses of             DNP--ASC and DNP.sub.3 --OA on days 0 and 28, respectively. The test mice      were pretreated with 1 mg of DNP.sub.2.4 --PVA.sub.10 on day -2.               **N.T. = not tested since the animals were not immunized with the              corresponding carriers.                                                  

                  TABLE IX                                                         ______________________________________                                         TIME OF TREATMENT OF SENSITIZED CELL                                           DONORS WITH DNP.sub.2.4PVA.sub.10                                              PRIOR TO ADOPTIVE CELL TRANSFER                                                 ##STR2##                                                                      TIME OF TREATMENT                                                              OF CELL DONORS     PCA TITERS**                                                PRIOR TO CELL TRANSFER*                                                                           ANTI-DNP   ANTI-OA                                          ______________________________________                                         5 hours            900        1,910                                            1 day              920        1,950                                            2 days             100        2,000                                            3 days             300        1,910                                            4 days              80        1,950                                            7 days              80        1,900                                            14 days             90        1,800                                            CONTROL MICE       1,430      2,400                                            ______________________________________                                          *Donors of spleen cells were preimmunized with a sensitizing dose of           DNP.sub.3OA on day -49. A single dose of 1 mg DNP.sub.2.4PVA.sub.10 was        injected i.p. into these donors at various times prior to transfer of          their spleen cells (3 × 10.sup.7) into Xirradiated (650 R)               recipients. These recipients were challenged within 4 hours with a             sensitizing dose of DNP.sub.3OA.                                               **The PCA titers were assayed 14 days after cell transfer.               

                                      TABLE X                                      __________________________________________________________________________     PREVENTION OF SYSTEMIC ANAPHYLAXIS BY TREATMENT OF                             SENSITIZED MICE WITH DNP.sub.2.4PVA.sub.10                                      ##STR3##                                                                                                            SYSTEMIC REACTION ON                            TREATMENT OF MICE PCA TITERS** CHALLENGE WITH                           GROUP  SENSITIZED ON DAY -21*                                                                           ANTI-DNP                                                                              ANTI-OA                                                                              DNP.sub.9M.sub.γ G                                                                OA                              __________________________________________________________________________     CONTROL                                                                               Day 0:                                                                              DNP.sub.3OA + Al(OH).sub.3                                                                  3,320  3,470 Death in 41, 44,                                                                        Death in 11, 11, 15,            (5 mice)    + B. pertussis            44, 47 and 49 min.                                                                      18 and 25 min.                         Day 7:                                                                              Challenge i.v. with                                                            2 mg DNP.sub.9M.sub.γ G or OA                                TEST   Day -1:                                                                             1 mg DNP.sub.2.4PVA.sub.10                                                                   <10   2,560 No symptoms                                                                             Death in 15, 18, 19,            (5 mice)                                                                              Day 0:                                                                              DNP.sub.3OA + Al(OH).sub.3                                                                               (5 mice) 19 and 21 min.                              + B. pertussis                                                            Day 7:                                                                              Challenge i.v. with                                                            2 mg DNP.sub.9M.sub.γ G or OA                                __________________________________________________________________________      *All mice received a sensitizing dose of DNP.sub.3OA + (Al(OH).sub.3 3         weeks before the experiment. Mice in Group I were resensitized on day 0        with 1 μg of DNP.sub.3OA suspended in 1 mg Al(OH).sub.3 and 8 ×       10.sup.9 B. pertussis. Mice in Group II received an additional i.p.            injection of 1 mg of DNP.sub.2.4PVA.sub.10 one day before resensitization      After 7 days, mice were bled and challenged i.v. with 2 mg of                  DNP.sub.9M.sub.γ G or OA, as indicated in the Table.                      **The PCA titers refer to the reaginic antibodies in sera of mice 7 days      after immunization.                                                      

LEGENDS TO FIGURES FIG. 1.--Suppression of the induction of anti-DNP IgE response

All mice received an i.p. sensitizing dose of 1 μg DNP₃ -OA in presence of 1 mg Al(OH)₃. Mice in test group received an additional i.p. injection of 1 mg DNP₃.4 -PVA₁₀ 7 days prior to sensitization. The IgE responses specific for the DNP determinant (Δ--Δ) and for the carrier (O--O) are expressed in terms of PCA titers.

FIG. 2.--Abrogation of an ongoing anti-DNP IgE response with DNP₃.4 -PVA₁₀

All mice were sensitized with 1 μg DNP₃ -OA in presence of 1 mg Al(OH)₃. Mice in the test group received an additional i.p. injection of 1 mg DNP₃.4 -PVA₁₀ 14 days after sensitization. The IgE responses specific for the DNP determinant and the carrier are represented by triangles and circles, respectively.

FIG. 3.--Active suppression of anti-DNP IgE response of primed cells with tolerized cells in adoptive transfer system

Different combinations of spleen cells from primed (S_(p)) and tolerized (S_(t)) mice, as indicated in the Fig., were transferred into X-irradiated (650 R) recipients. The primed donors were sensitized with DNP₃ -OA 30 days before the cell transfer, whereas the tolerized donors received only a single injection of 1 mg DNP₂.4 -PVA₁₀ 7 days prior to cell transfer. The IgE responses to both the hapten and the carrier protein, represented by the hatched and open bars, respectively, were measured in terms of PCA titers 14 days after cell transfer.

CONCLUSIONS

From the above, it may be concluded that the PVA conjugates described in the present invention suppress the formation of IgE antibodies to the appropriate haptens and that the mechanism underlying this suppression may involve the production of suppressor cells, which interfere actively with the corresponding IgE-producing lymphoid cells.

It will be appreciated by those skilled in the art that the treatment of humans with BPO-PVA conjugates prepared as described in the present invention differs significantly from currently available therapeutic procedures in several respects. To begin with, the conjugate will be used in the treatment of humans who already are sensitive to penicillin (and thus the sensitizing step(s) for induction of sensitivity to BPO, which was used in experimental animals, will not be applicable). In view of the fact that injection of polyvalent BPO conjugates into allergic individuals may lead to typical allergic manifestations due to the release of histamine and other vasoactive compounds from their mast cells and basophils, it is visualized that a therapeutic regimen for the immunosuppression of anti-BPO IgE antibodies in man would involve the administration of conjugates of BPO and the simultaneous treatment of the patients with anti-allergic drugs. In this connection, it is interesting to note in Table IV that conjugates with very low epitope density, such as BPO₁.1 -PVA, were still highly effective in suppressing the anti-BPO IgE response. Moreover, it has been recently found that the tolerogenic BPO₁.1 -PVA conjugate elicited only very weak PCA reactions in rats sensitized intradermally with mouse anti-BPO reaginic antibodies. Hence, the possibility of eliciting systemic reactions in allergic patients on treatment with tolerogenic conjugates might be minimized or eliminated by the use of PVA conjugates with low epitope density. In this respect, the BPO-PVA conjugates described in this invention have superior therapeutic values over other tolerogenic conjugates, such as BPO-isologous γ-globulins or BPO-D-GL, which required high epitope density in order to be tolerogenic.

On the basis of the evidence presented for both the DNP and BPO IgE antibody systems, it may also be postulated that this treatment could be extended to the suppression of allergies to other antibiotic molecules by administration of conjugates consisting of PVA coupled to the antilbiotic molecules in question. 

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
 1. A process for the preparation of nonimmunogenic conjugates, suitable for suppressing the formation of reaginic antibodies to penicillin (BPO) in a host, the said conjugate being prepared by the reaction of penicillin G in the presence of a suitable salt, at alkaline pH, with an amino derivative of the water-soluble, nonimmunogenic polymer, polyvinyl alcohol (PVA).
 2. A process as stated in claim 1, wherein the amino derivative of PVA is prepared by reacting 1,6-hexanediamine with polyvinyl alcohols which had been activated with cyanogen bromide at a pH range of 9-11.
 3. A process as stated in claim 1, wherein the polyvinyl alcohol has a molecular weight in the range of 10,000 to 14,000.
 4. A process as stated in claim 1, in which the suitable salts are selected from the group consisting of potassium carbonate and sodium carbonate.
 5. A process as stated in claim 1, in which the alkaline pH had a range of 8-11.
 6. A conjugate consisting of BPO groups coupled to polyvinyl alcohol, suitable for suppressing the formation of reaginic antibodies to penicillin in an allergic host, the conjugate containing an average of 0.7 to 3.9 BPO groups per molecule of polyvinyl alcohol, the said conjugate being prepared according to the process of claim
 1. 7. A process according to claim 1 wherein said alkaline pH is 9.8. 